The overall objective of our work is to understand the mechanisms of glomerular endothelial (GEn) cell differentiation. The current proposal explores the role of podocytes in this process, podocytes being the immediate neighbors of GEn cells in vivo. In mature glomeruli, podocytes express extraordinarily high levels of the angiogenic growth factor VEGF, yet GEn cells in vivo ordinarily do not proliferate and do not undergo angiogenesis. indeed, even in diabetic patients with retJnopathy and nephropathy, retinal angiogenesis is stimulated but glomerular angiogenesis is not observed. We propose that podoctye derived signals are instrumental in locking the GEn cells into a terminally differentiated state. We find that in vitro GEn-podocyte co-culture using monolayer and trans-filter assays, results in the inhibition of GEn migration, and strong protection of GEn cells from apoptosis. Hence, podocytes, in co-culture with GEn cells produce soluble anti-apoptotic, anti-chemokinetic mediators. We know that podocytes produce two relatively endothelial cell specific survival factors; angiopoietin-1 and VEGF-A. We will therefore explore the hypothesis that angiopoietin-1 and/or VEGF-A serve survival and differentiation functions for GEn cells in vitro and in vivo. Both, angiopoietin-1 and VEGF stimulate endothelial cell migration in vitro, but differentiated podocytes inhibit GEn migration. Inhibition of VEGF- stimulated migration by endothelial cells is typically seen when metalloproteinase activity is blocked. In this regard, we find that GEn ceils express abundant collagen XVIII, and generate soluble endostatin-Ilike collagen XVlli NCl domain cleavage products, likely through the actin of matrix metalloproteinases. Podocyte co-culture completely abrogated production of the endostatinlike collagen XVill fragments. We therefore propose to test the hypothesis that podocytes inhibit matrix meta[ioproteinase activity, and consequently suppress the angiogenic phenotype of GEn cells. We propose to use GEn cells and podocytes in culture to define the potential role of three putative podocyte derived mediators, VEGF-A, angiopoietin-1 and metalloproteinase inhibitors in GEn differentiation. In addition, using conventional light and electron microscopy, as well as intravitai microscopy, we will explore the role of these podocyte derived stimuli in the process of glomerular capillary differentiation in neonatal mice in vivo.